Grain compositions containing pre-biotic isomalto-oligosaccharides and methods of making and using same

ABSTRACT

Methods for the production of substrate, tuber, and grain compositions containing isomalto-oligosaccharides are described. The methods comprise (a) contacting a substrate, tuber or grain containing ungelatinized starch with a maltogenic enzyme and a starch liquefying enzyme to produce maltose; (b) contacting said maltose with a transglucosidic enzyme, wherein said steps (a) and step (b) occur at a temperature less than or at a starch gelatinization temperature; and (c) obtaining a substrate, grain or tuber composition having an enzymatically produced isomalto-oligosaccharide, wherein the oligosaccharide is derived from the grain. The maltogenic enzyme can be either exogenous or endogenous to the grain. The contacting steps can be sequential or concurrent. The present invention also describes flour, oral rehydrating solutions, beer adjuncts, food, feed, beverage additives incorporating the grain compositions made as described.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/477,199, filed Jun. 9, 2003, and U.S. Provisional Application No.60/453,668, filed Mar. 10, 2003.

TECHNICAL FIELD

The present invention describes grain compositions containingisomalto-oligosaccharides and methods for making the same. The methodincludes the derivation of the isomalto-oligosaccharides from the starchcontained within the grain.

BACKGROUND OF THE INVENTION

Isomalto-oligosaccharides (“IMOs”) are mixed linkage oligosaccharides,having mixtures of 1,4 alpha and/or 1,6 alpha glucosidic linkages. Theyare also known as anomalously linked oligosaccharides (“ALOs”).Isomalto-oligosaccharides contain a substantial amount of branchedoligo-saccharides such as isomaltose, panose, isomaltotriose,isomaltotetraose, isopanose and higher branched oligo-saccharides.

There is a market demand for products containing IMO's. IMO products aresold in powder or liquid form, depending on the intended application.The potential applications are situated in the food area. Examples ofIMO products are: seasonings (mayonnaise, vinegar, soup base etc.),confectionery (candy, chewing gum, chocolate, ice cream, sherbet,syrup), processed foods of fruits and vegetables (jam, marmalade, fruitsauces, pickles), meat or fish foods (ham, sausage, etc.), bakeryproducts (bread, cake, cookie, pastry), precooked foods (salad, boiledbeans, etc.), canned and bottled foods, convenience foods (instantcoffee, instant cake base, etc.), and beverages, both alcoholic (liquor,seju, wine, sake, beer [International Publication No. WO 02/20712 A1],etc.) and non-alcoholic (coffee, juice, nectar, aerated or carbonateddrinks, lemonade, cola). Isomalto-oligosaccharide can further be appliedas ingredients in animal feed and pet foods. Non-food application areasare cosmetics and medicine (cigarette, lipstick, toothpaste, internalmedicine, etc.).

Isomalto-oligosaccharides belong to a group of oligosaccharidesclassified as functional-health food oligosaccharides (“FHFO”).Exemplary IMO's include fructo-oligosaccharides,galacto-oligosaccharides, xylo-oligosaccharides andgentio-oligosaccharides. IMO's have been linked to the increase of thegeneral well being of humans and animals when taken orally on a regulardaily basis and are classified as “prebiotics”. Prebiotics are definedas non-digestible substances (e.g., dietary fiber) that exert somebiological effect on humans by selective stimulation of growth orbioactivity of beneficial microorganisms either present ortherapeutically introduced to the intestine. (Przemyslaw Jan Tomasik andPiotr Tomasik. 2003 American Association of Cereal Chemists, Inc. 80(2):113-117). The “prebiotic” action of the oligosaccharides is to increasethe numbers of bifidobacteria and lactobacilli (“prebiotic”) in thelarge intestine and to reduce the concentration of putrifactivebacteria. Bifidobacteria are associated with some health promotingproperties like the inhibition of the growth of pathogens, either byacid formation or by anti-microbial activity. They are also associatedwith such diverse effects as the modulation of the immune system(anti-tumor properties), the reduction of the levels of triglyceridesand cholesterol, the production of vitamins (B group), the reduction ofblood ammonia concentrations, the prevention of translocation, therestoration of the normal gut flora after anti-microbial therapy, theproduction of digestive enzymes, the reduction of antibiotic associatedside effects (Kohmoto T., Fukui F., Takaku H., Machida Y., et al.,Bifidobacteria Microflora, 7(2)(1988),61-69; Kohmoto K., Tsuji K.,Kaneko T., Shiota M., et al., Biosc. Biotech. Biochem.,56(6)(1992),937-940; Kaneko T., Kohmoto T., Kikuchi H., Fukui F., etal., Nippon Nogeikagaku Kaishi, 66(8)(1992),1211-1220, Park J-H,Jin-Young Y., Ok-Ho S., Hyun-Kyung S., et al., Kor. J. Appl. Microbiol.Biotechnol., 20(3)(1992), 237-242). Modler, H. W., 1992, “Compoundswhich enhance the growth of Prebiotic Bacteria”, presented at theInternational Roundtable on Animal Feed Biotechnology, Ottawa, Ontario,Canada.

The isomalto-oligosaccharides are synthesized by an enzyme catalyzedtransglucosylation reaction using a D-glucosyltransferase (E.C.2.4.1.24, transglucosidase, alpha-glucosidase). This enzyme catalyzesboth hydrolytic and transfer reactions on incubation withalpha-D-gluco-oligosaccharides. The transfer occurs most frequently to6-OH (hydroxyl group 6 of the glucose molecule), producing isomaltosefrom D-glucose, or panose from maltose. The enzyme can also transfer tothe 2-OH or 3-OH of D-glucose to form kojibiose or nigerose, or back to4-OH to reform maltose. As a result of transglucosidase reactions, themalto-oligosaccharides are converted into isomalto-oligosaccharidesresulting in a class of oligosaccharides containing a higher proportionof glucose moieties linked to a primary hydroxyl group of a glucosemolecule from the non-reducing end, e.g., by alpha-D-1,6 glucosidiclinkages. The transglucosidase from A. niger acts only onoligosaccharides with a low degree polymerization (DP) (McCleary B. V.,Gibson T. S., Carbohydrate Research 185(1989)147-162; Benson C. P.,Kelly C. T., Fogarty W. M., J. Chem. Tech. Biotechnol., 32(1982)790-798;Pazur J. H., Tominaga Y., DeBrosse C. W., Jackman L. M., CarbohydrateResearch, 61(1978) 279-290). Degree of polymerization refers to thenumber of dextrose units. For example, a di-glucosyl molecule, forexample maltose, has a DP of 2. These sugars are receiving increasedattention as food additives because they help prevent dental caries(Oshima, et. al 1988. The caries inhibitory effects of gos-sugar invitro and rat experiments. Microbial Immunol. 32, 1093-1105) and improvehuman intestinal microflora acting as a growth factor (prebiotic) forbifidobacteria (Komoto, et. al 1988; Effect of Isomalto-oligosaccharideson human fecal flora Bifidobacteria Micro flora 7, 61-69).

Isomalto-oligosaccharides can be obtained in different ways. For exampleglucose syrups at high dry solids concentration i.e. 60-80% are treatedwith glucoamylase resulting in the formation ofisomalto-oligosaccharides mainly DP2. The high solids levels are presentto force the reaction to reverse from the normal direction in favor ofhydrolysis.

Grains, including wheat, barley, etc., are excellent raw materials inthe commercial production of many value added functional foodingredients such as wheat flour, starch, starch hydrolysates (glucose,fructose, high maltose syrup, etc.) and wheat gluten. Syrup containing ahigh level of maltose is also used in many microbial fermentations as acarbon source in the production of antibiotics, pharmaceuticals,vaccines, biochemical, such as alcohol (both potable and fuel), aminoacids, organic acids, etc and recently in the production of functionalhealth-food oligosaccarides called isomalto-oligosaccharides. In aconventional process for the production of starch hydrolysate, such asmaltose syrups, the insoluble granular starch is generally separatedfrom other cellular components of wheat prior to the hydrolysis bystarch liquefying and maltogenic alpha amylases enzymes. Maltose is adisaccharide consisting of two glucosyl residues linked by α 1-4D-glucosidic linkage and is the smallest in the family ofmalto-oligosaccharides. It is produced on a large scale as syrup, powderand crystals in several grades of purity. Various maltose syrups aredrawing considerable interest for commercial applications in brewing,baking, soft drink canning, confectionary and other food and beverageindustries. Ultra pure maltose is used as an intravenous nutrient inJapan. Catalytic reduction of maltose results in maltitol, which isconsidered to be a low calorie sweetener. Recently, high maltose syruphas become a key raw material for industrial production ofisomalto-oligosaccharides (J. K. Shetty and O. J. Lantero, 1999“Transglucosylation of Malto-oligosaccharides.” Paper presented at 50thStarch Convention, Detmold, Germany).

In a conventional process for the production of starch hydrolysate suchas high maltose syrup, the insoluble starch is separated prior to thehydrolysis by thermostable liquefying alpha amylases [EC 3.2.1.2,alpha(1,4)-glucan glucanohydrolase] derived either from Bacilluslicheniformis or Bacillus stearothermophilus. Hydrolysis of the purifiedstarch (refined) is carried out by suspending insoluble granular starchin water (30-35% dissolved solid basis [dsb]) and heated to atemperature of between 85° C. and 120° C. to solubilize the starch andmaking it susceptible for enzymatic hydrolysis. The liquefied starch isfurther processed to manufacture starch hydrolysate with differentcarbohydrate composition using specific maltose producing enzymes suchas fungal alpha amylase (sold under the tradename CLARASE L fromGenencor International, Palo Alto, Calif.) for syrup containing lessthan 55% maltose, β Amylase (sold under the tradename OPTIMALT BBA fromGenencor International, Palo Alto, Calif.) for syrup containing maltosecontent between 55% and 62% and less than 1% glucose. For higher levelsof maltose syrup, >62%, addition of debranching enzyme (sold under thetradename OPTIMAX L-1000 from Genencor International, Palo Alto, Calif.)in conjunction β Amylase is useful. (Faigh, J.; Duan, G.; Strohm, B. andShetty, J. (2002) “Production of Maltose, High Maltose & Very HighMaltose Syrups,” Technical Bulletin, Genencor International Inc.).

A process for converting granular starch (refined) into solublehydrolysate by incubating with bacterial alpha amylase at a temperaturebelow the starch gelatinization temperature (Leach et. al 1978; U.S.Pat. No. 4,113,509) and subsequent hydrolysis by beta amylase to producehigh maltose syrup have been reported (Leach et. al 1975; U.S. Pat. No.3,922,196), However the syrup produced by such process resulted in only55% maltose of the total sugar content, with a very high level ofmaltotriose. The process for producing high maltose syrup usingliquefied starch (gelatinized followed by hydrolysis using thermostablealpha amylase) is described in European Patent Application #0905256(Christophersen, et. al 2000) and U.S. Pat. No. 5,141,859 (Nimmi, et. al1992). The process is cumbersome, expensive and it requires theseparation of starch from other cellular components, high cost of theadditional maltose producing enzymes, high temperature treatment andlonger reaction time. European Patent Application #0350737 A2 (Shinke,et. al 1989) disclosed a process for producing maltose syrup byhydrolyzing a granular (purified) starch from corn, wheat, potato andsweet potato at 60° C. without the conventional liquefaction step(gelatinization followed by liquefaction at high temperature) using analpha amylase from Bacillus stearothermophilus. However, the hydrolyzedstarch resulted in a maltose concentration ranging from 50% to 55%. Thesyrup also contained very high level of maltotriose (30-36%). Theprocess resulted in a ratio of maltose to maltotriose less than 2.0irrespective of the source of the starch. Maltose syrup containing ahigh level of maltotriose is not a preferred substrate as carbon feed inmany microbial fermentations including the alcohol fermentation by yeastbecause of the difficulties in metabolizing maltotriose. Maltose is apreferred donor of glucosyl residue in the transglucosylation reactioncatalyzed by glucosyltransferases in the production ofisomalto-oligosaccharides (J. K. Shetty and O. J. Lantero, 1999“Transglucosylation of Malto-oligosaccharides.” Paper presented at 50thStarch Convention, Detmold, Germany). U.S. Pat. No. 6,361,809 describeda method for producing maltose and a limit dextrin by treating thepurified granular waxy maize starch with a hydrolase, maltogenase alphaamylase classified as EC 3.2.1.133 from Bacillus stearothermophilusfollowed by separating the maltose using ultra filtration process.Evaporation of the dilute permeate containing the maltose is expensivebecause of high energy cost and also faces a very high risk of microbialcontamination.

Traditionally grains such as wheat, malt, sorghum (milo), millet (ragi),particularly whole grains are used in nutrition as carriers of macro-and micro-elements, proteins, fiber and vitamins. The majority of cerealgrains appeared to be too readily digested to play an effective role asprebiotics or even as nutraceuticals. It has been suggested thatdesigning genetically modified, less digestible cereals suitable asprebiotics to manipulate gut microflora (Gibson, G. R, and Roberfroid,M. B. 1995, Dietary modulation of the human colonic microflora:Introducing the concept of prebiotics. J. Nutr. 125, 1401-1412).

There is a continuing interest in methods for producing graincompositions with isomalto-oligosaccharides enzymatically derived fromthe source substrate, e.g., grain or tuber, without having to separatethe starch from other grain components and/or subject the starch of thesubstrate to high temperatures of jet cooking prior totransglucosidation action. There is also a continuing interest in low pHprocesses for minimizing the risk of microbial contamination. Thepresent invention addresses these interests.

SUMMARY OF THE INVENTION

The present invention describes a method for making anisomalto-oligosaccharide grain composition said method comprising: (a)contacting a ungelatinized starch containing grain with a maltogenicenzyme and a starch liquefying enzyme to produce maltose; (b) contactingsaid maltose with a transglucosidic enzyme, wherein said steps (a) andstep (b) occur at a temperature less than or at a starch gelatinizationtemperature; and (c) obtaining a grain composition having anenzymatically produced isomalto oligosaccharide, wherein saidoligosaccharide is derived from said grain.

Optionally, in one embodiment, the steps (a) and (b) occur concurrently.In another embodiment, the method further includes a step of drying saidgrain composition. In another embodiment the grain is selected from thegroup consisting of wheat, rye, barley, malt and rice. In anotherembodiment the grain is selected from the group consisting of sorghum,millet and rice. In another embodiment, the maltogenic enzyme is a betaamylase. In another embodiment, the maltogenic enzyme is endogenous tosaid grain. In another embodiment, the maltogenic enzyme is exogenous tosaid grain. In another embodiment, the starch liquefying enzyme is analpha amylase derived from a Bacillus. In another embodiment, the starchliquefying enzyme is derived from Bacillus licheniformis or Bacillusstearothermophilus. In another embodiment, the transglucosidic enzyme isa transglucosidase. In another embodiment, the transglucosidase isderived from Aspergillus. In another embodiment, the transglucosidasederived from Aspergillus niger. Another embodiment of the presentinvention includes a grain composition produced according to abovedescribed method. Another embodiment of the present invention includes afood additive comprising said grain composition described above.

The present invention also describes a method for making anisomalto-oligosaccharides enriched flours at temperatures at or belowthe gelatinization temperature wherein an ungelatinized grain having anendogenous maltogenic enzyme are contacted with a solubilizing enzymeselected from Bacillus to produce a maltose syrup. The maltose syrup iscontacted with a transglucosidase to produce a substrate (tuber orgrain) composition including isomalto-saccharides derived therefrom.

The present invention also describes a method for making anisomalto-oligosaccharides enriched flours at temperatures at or belowthe gelatinization temperature wherein an ungelatinized grain having anendogenous maltogenic enzyme (wheat, barley, etc.) are mixed withungelatinized grain not having endogenous maltogenic enzymes (e.g.,sorghum, miller or rice), the grain mixture being contacted with asolubilizing enzyme selected from Bacillus to produce a maltose syrup.The maltose syrup is contacted with a transglucosidase to produce asubstrate (tuber or grain) composition including isomalto-saccharidesderived therefrom.

The present invention also describes a method for making a wheat graincomposition said method comprising: (a) contacting an ungelatinizedwheat grain having an endogenous maltogenic beta-amylase and a starchliquefying alpha amylase from Bacillus to produce maltose; (b)contacting said maltose with a transglucosidase, wherein said steps (a)and step (b) occur at a temperature less than wheat gelatinizingtemperature; and (c) obtaining a wheat grain composition having anenzymatically produced isomalto-oligosaccharide, wherein saidoligosaccharide is derived from said ungelatinized grain.

Optionally, in another embodiment the method uses the above method formaking a grain composition for making a food additive. Anotherembodiment includes a grain composition made accordingly. Anotherembodiment includes a flour comprising the grain composition describedabove. Another embodiment includes an isomalto-saccharide made accordingto the method described above. Another embodiment includes an oralrehydration solution comprising the isomalto-oligosaccharide above.Another embodiment includes a grain composition comprising anungelatinized grain and at least one isomalto-oligosaccharide, whereinsaid isomalto-oligosaccharide is enzymatically derived from saidungelatinized grain. In another embodiment the grain compositioncontains greater than 1% by weight of at least oneisomalto-oligosaccharide.

DESCRIPTION OF THE FIGURES

FIG. 1 is a flowchart describing the production ofisomalto-oligosaccharide enriched flour.

FIG. 2 is another flowchart describing the production ofisomalto-oligosaccharide enriched wheat flour.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “grain” refers to a plant, which is classified as a cereal oras a monocotyledonous plant belonging to the Poales order, in particularthe family Poaceae. Examples of plants belonging thereof are plantsselected from the genuses Triticum (wheat), Hordeum (barley); Secale(rye); Zea (corn or maize); Avena (oats), Fagopryum (buckwheat); Sorghum(sorghum or milo), Panicum or Setaria (millet or ragi); or Oryza (rice).

For example, in one embodiment, the term “wheat” refers to a plant whichis classified or once was classified as a strain of Triticum aestivum.

For example, in one embodiment, the term “barley” refers to a plantwhich is classified or once was classified as a strain of Hordeumvulgare.

For example, in one embodiment, the term “rye” refers to a plant, whichis classified or once was classified as a strain of Secale cereale.

For example, in one embodiment, the term “corn” refers to a plant, whichis classified or once was classified as a strain of Zea mays.

For example, in one embodiment, the term “oats” refers to a plant, whichis classified or once was classified as a strain of Avena sativa.

For example, in one embodiment, the term “buckwheat” refers to a plant,which is classified or once was classified as a strain of Fagopryumesculentum.

For example, in one embodiment, the term “sorghum” refers to a plant,which is classified or once was classified as a strain of Sorghumbicolor.

For example, in one embodiment, the term “millet” refers to a plant,which is classified or once was classified as a strain of Panicummiliaceum or Setaria italica.

For example, in one embodiment, the term “rice” refers to a plant whichis classified or once was classified as a strain of Oryza sativa.

The term tuber refers to a starchy storage organ (for example a potato,sweet potato, yam, manioc, etc) formed by swelling of an undergroundstem or the distal end of a root.

For example, in one embodiment, the term “potato” refers to a plantwhich is classified or once was classified as a strain of Solanumtuberosum.

For example, in one embodiment, the term “sweet potato” refers to aplant which is classified or once was classified as a strain ofIpom[oe]a balatas.

For example, in one embodiment, the term “yam” refers to a plant whichis classified or once was classified as a strain of Dioscorea sativa, D.villosa, C. batatas.

The term substrate refers to materials that can be enzymaticallyconverted to maltose and thus IMO's. The term “substrate” includes, forexample, grains and tubers. Furthermore, the term substrate includes allforms of the grain (polished or unpolished) or tuber, such as wholegrains, broken grains, grits and flour and any plant part.

The term “starch” refers to any material comprised of the complexpolysaccharide carbohydrates of plants, comprised of amylose andamylopectin with the formula (C₆H₁₀O₅)_(x), wherein X can be any number.

The term “granular starch” refers to uncooked (raw) starch, which hasnot been subject to gelatinization. The term “gelatinization” refers tosolubilization of a starch molecule to form a viscous suspension.

The phrases “substrate”, “grain” or “tuber” containing ungelatinizedstarch” refer to an ungelatinized substrate, grain or tuber that is notsubjected to temperatures greater than the starch gelatinizationtemperatures which result in effecting a gelatinization or liquefactionof the starch contained within the substrate.

The term “maltose” refers to a disaccharide having two glucosyl residueslinked by an alpha 1-4 D-glucosidic linkage

The term “isomaltose” refers to a disaccharide having two glucosylresidues linked by an alpha 1,6 D-glucosidic linkage.

The term “isomalto-oligosaccharide” (IMO) refers to sugars having atleast two glucosyl residues linked by alpha 1,6 glucosidic linkages atthe non-reducing end. In addition term refers to anomalously linkedoligosaccharides, saccharides having both alpha 1,6 and alpha 1,4glucosidic linkages. Exemplary isomalto-oligosaccharides includeisomaltose, panose, and isomalto-triose The term“isomalto-oligosaccharide” grain composition refers to graincompositions characterized by isomalto-sugars level of at least 1% (w/w%) of the total sugar content as determined by high performance liquidchromatographic methods.

The term “maltogenic enzyme” refers to an enzyme that converts starch tomaltose. Exemplary maltogenic enzymes include fungal, bacterial andplant derived alpha amylases and beta-amylases.

The term “amylases” refers to enzymes that catalyze the hydrolysis ofstarches.

The term “alpha-amylase” refers to enzymes of the class (E.C.) 3.2.1.1and/or 3.2.1.133 that catalyze the hydrolysis of alpha-1,4-glucosidiclinkages. These enzymes have also been described as those effecting theexo or endohydrolysis of 1,4-alpha-D-glucosidic linkages inpolysaccharides containing 1,4-alpha-linked D-glucose units. Anotherterm used to describe these enzymes is glycogenase. Exemplary enzymesinclude alpha-1,4-glucan 4-glucanohydrase glucanohydrolase.

The term “beta-amylase” refers to enzymes of the class (E.C.) 3.2.1.2that catalyze the hydrolysis of alpha-1,4 glucosidic linkages releasingmaltose units. These enzymes have also been described as those effectingthe hydrolysis of 1,4-alpha-D-glucosidic linkages in polysaccharides soas to remove successive maltose units from the non-reducing end ofchains.

The term “transglucosidic” enzyme refers to an enzyme that catalyzesboth hydrolytic and transfer reactions in incubation with alphaD-gluco-oligosaccharides. Exemplary enzymes include transglucosidasesand/or those of the class (E.C.) 2.4.1.24, e.g., D-glucosyltransferase.These enzymes have also been referred to as 1,4-alpha-glucan6-alpha-glucosyltransferase and oligoglucan-branchingglycosyltransferase.

The term debranching enzyme refers to enzymes that catalyze thehydrolysis of alpha-1,6-linkages. An enzyme of the class E.C. 3.2.1.41is useful in this regard. An exemplary enzyme of this class is apullanase, also known as alpha-dextrin endo-1,6-alpha glucosidase, limitdextrinase, debranching enzyme, amylopectin 1,6-glucanohydrolase.Additional exemplary enzymes of the class (E.C.) 3.2.1.41, e.g.,pullulanases, [alpha-(1-6)-glucan 6-glucanohydrolase, also calledalpha-(1,6)-glucosidase]).

The term “starch gelatinizing temperatures” refers to a temperaturesufficiently high to effect liquefying or gelatinization of granularstarch. Heating a starch in water causes the starch granules to swell.At sufficient solids concentration, the swollen granules occupy most ofthe space and a viscous mass, called a paste, results. Solubilization ofstarch molecule is called gelatinization. Gelatinization is accompaniedby a loss of birefringence. The term starch gelatinizing temperaturerefers to the temperature at which gelatinization occurs.

The term “starch liquefying enzyme” refers to an enzyme that effects thefluidization of granular starch. Exemplary starch liquefying enzymesinclude alpha amylases of the class (E.C.) 3.2.1.1.

The term “endogenous” refers to the enzyme being present in the grain ortuber without having to resort to adding the maltogenic enzyme to thegrain.

The term “exogenous” enzyme refers to an enzyme that is not presentwithin the grain. Exemplary exogenous enzymes include, for example,maltogenic enzymes not present in the wild-type substrate, e.g., rice,millet, etc.

The term “total sugar content” refers to the total amount of sugarpresent in a starch, grain or tuber composition.

The term “IMO No.” is calculated as the sum of isomaltose, panaose,isomaltotriose and branched sugars greater than DP3. The IMO Numberprovides an indication of the amount of IMO compounds present in thecompound or solution.

The term “ratio of branched sugars” (“RBS”) refers to the ratio ofmaltose (DP2) present in the grain as compared to the level ofmaltotriose (DP3) present in the resultant grain composition.

The term “Degrees of Diastatic Power” (DP°) unit refers to the amount ofenzyme contained in 0.10 ml of a 5% solution of the sample enzymepreparation that will produce sufficient reducing sugars to reduce 5 mlof Fehling's solution when the sample is incubated with 100 ml ofsubstrate for 1 hour at 20° C. (68° F.).

The term “DE” or “dextrose equivalent” is an industry standard formeasuring the concentration of total reducing sugars, calculated asD-glucose on a dry weight basis. Unhydrolyzed granular starch has a DEthat is essentially 0 and D-glucose has a DE of 100.

The term “total sugar content” refers to the total sugar content presentin a starch composition.

The terms “dry solid basis” and “dsb” refer to the total amount ofcompound, e.g., flour, of a slurry (in %) on a dry weight basis.

The terms “dry solid content”, “dry solid granular starch”, “dry solidstarch” and “(dss)” refer to the total starch of a slurry (in %) on adry weight basis.

The term “Brix” refers to a well known hydrometer scale for measuringthe sugar content of a solution at a given temperature. Thus the term“Brix” refers to a measure of the solubilized sugars in solution. TheBrix scale measures the number of grams of sucrose present per 100 gramsof aqueous sugar solution (the total solubilized solid content). Forexample, a measurement of 1.00 Brix refers to about 10 mg/ml of sugar insolution. Brix measurements are frequently made by use of a hydrometeror refractometer.

The term “degree of polymerization (DP)” refers to the number (n) ofanhydroglucopyranose units in a given saccharide. Examples of DP1 arethe monosaccharides, such as glucose and fructose. Examples of DP2 arethe disaccharides, such as maltose and sucrose. A “DP4+” denotespolymers with a degree of polymerization of greater than 3.

The term “enzymatically produced” refers to enzymatic catalysis of thesubstrate to the IMO as opposed to chemical or organic chemicalsynthesis of the IMO. The term “filamentous fungi” refers to allfilamentous forms of the subdivision Eumycotina (See, Alexopoulos, C. J.(1962), INTRODUCTORY MYCOLOGY, New York: Wiley). These fungi arecharacterized by a vegetative mycelium with a cell wall composed ofchitin, cellulose, and other complex polysaccharides. The filamentousfungi of the present invention are morphologically, physiologically, andgenetically distinct from yeasts. Vegetative growth by filamentous fungiis by hyphal elongation and carbon catabolism is obligatory aerobic. Inthe present invention, the filamentous fungal parent cell may be a cellof a species of, but not limited to, Trichoderma, e.g., Trichodermareesei (previously classified as T. longibrachiatum and currently alsoknown as Hypocrea jecorina), Trichoderma viride, Trichoderma koningii,Trichoderma harzianum; Penicillium sp.; Humicola sp., including Humicolainsolens and Humicola grisea; Chrysosporium sp., including C.lucknowense; Gliocladium sp.; Aspergillus sp., including A. oryzae, A.nidulans, A. niger, and A. awamori; Fusarium sp., Neurospora sp.,Hypocrea sp., and Emericella sp. Reference is also made to Innis et al.,(1985)

Sci. 228:21-26.

The term “Aspergillus” or “Aspergillus sp.” refers to any fungal strain,which had previously been classified as Asperilglus or is currentlyclassified as Aspergillus.

The term “bacterial” refers to Bacillus species of, but not limited toB. subtilis, B. amyloliquefaciesn, B. lentus, B. Carlsberg, B.licheniformis, and B. stearothermophilus

The term “plant origin” refers to the enzyme being derived, extracted,isolated, expressed from a plant source, for example from barley malt,soybean, wheat or barley.

The term “contacting” refers to the placing of the respective enzyme[s]in sufficiently close proximity to the respective substrate to enablethe enzyme[s] to convert the substrate to the desired end-product. Thoseskilled in the art will recognize that mixing solutions of the enzyme orenzymes with the respective substrates can effect contacting.

The term “incubating” refers to mixing a substrate containing substratewith the respective enzymes, e.g., liquefying or maltogenic ortransglucosidase under given conditions for a defined period of time.

The term “enzymatic conversion” refers to the modification of a ricesubstrate to yield soluble hydrolyzed granular rice starch andpreferably to yield glucose.

The term “slurry” refers to an aqueous mixture containing insolublegranular starch. Sometimes the terms “slurry” and “suspension” are usedinterchangeably herein.

The term “culturing” refers to growing a population of microbial cellsunder suitable conditions in a liquid or solid medium. In oneembodiment, culturing refers to fermentative bioconversion of a granularstarch substrate to glucose syrup or other desired end products(typically in a vessel or reactor). For example, in one embodiment, theterm alpha amylase enzyme unit is defined as the amount of alpha amylasewhich hydrolyzes 1 micromole of starch substrate in 1 min under standardassay conditions of pH 5.2 and 40° C. For example, in one embodiment,the term beta amylase enzyme unit is defined as the amount of betaamylase which hydrolyzes 1 micromole of starch substrate in 1 min understandard assay conditions of pH 4.6 and 20° C.

For example, in one embodiment, the term transglucosidase unit isdefined as the amount of transglucosidase which converts 1 micromole ofmaltose substrate in 1 min under standard assay conditions of pH 4.8 and37° C. In another embodiment, the term transglucosidase unit is definedas the amount of transglucosidase which produces 1 micromole of panoseper minute under standard assay conditions of pH 4.8 and 37° C.

For example, in one embodiment, the term one Liquefon Unit (LU) is themeasure of digestion time required to produce a color change with iodinesolution, indicating a definite stage of dextrinization of starchsubstrate under standard assay conditions of pH 5.6 and 25° C.

“ATCC” refers to American Type Culture Collection located at Manassas,Va. 20108 (ATCC, refer to the website for atcc.org).

“NRRL” refers to the Agricultural Research Service Culture Collection,National Center for Agricultural Utilization Research (and previouslyknown as USDA Northern Regional Research Laboratory), Peoria, Ill.

“NCBI” refers to the National Center for Biotechnology Information, NatlLibrary Med. (refer to the website for NCBI at the National institutesof Health ncbi.nlm.nih.gov/).

“A”, “an” and “the” include plural references unless the context clearlydictates otherwise.

The present invention describes a method for making anisomalto-oligosaccharide substrate, grain or tuber composition saidmethod comprising: (a) contacting a ungelatinized starch containingsubstrate, e.g., a grain or a tuber, with a maltogenic enzyme and astarch liquefying enzyme to produce maltose; (b) contacting said maltosewith a transglucosidic enzyme, wherein said steps (a) and step (b) occurat a temperature less than or at a starch gelatinization temperature;and (c) obtaining a substrate, grain or tuber composition having anenzymatically produced isomalto-oligosaccharide, wherein saidoligosaccharide is derived from said substrate, grain or tuber. Anembodiment of the present invention is depicted in FIG. 1.

The present invention also describes a method for making anisomalto-oligosaccharide-enriched substrate, grain or tubercompositions, flours, oral rehydrating solutions, and/or food additives,at temperatures at or below the gelatinization temperature wherein asubstrate having or containing an ungelatinized starch and havingendogenous maltogenic enzyme are contacted with a solubilizing enzymeselected from Bacillus to produce a maltose syrup. The maltose syrup isthen contacted with a transglucosidase at a temperature at or less thangelatinization or liquefaction temperatures to produce a graincomposition having isomalto-oligosaccharides. In one embodiment, thegrain composition is characterized by a sugar composition of greaterthan 60% maltose and a ratio of branched sugars of greater than 2.0 to1.0. The conversion of the substrate to the IMO can be enzymaticallyproduced.

The present invention also describes a method for making anisomalto-oligosaccharide substrate, grain or tuber composition, themethod comprising: (a) contacting a substrate, grain or tuber containinga starch with a maltogenic enzyme and a starch liquefying enzyme toproduce a maltose; (b) contacting the maltose with a transglucosidicenzyme, wherein the steps (a) and step (b) occur at a temperature lessthan or at starch gelatinization temperature; and (c) obtaining asubstrate, grain or tuber composition having an enzymatically producedisomalto-oligosaccharide, wherein the oligosaccharide is derived fromthe substrate, grain or tuber. The invention optionally furtherdescribes an additional step of separating soluble constituents frominsoluble constituents. The invention further describes an additionalstep of drying the substrate, grain or tuber composition. In oneembodiment the grain is selected from the group consisting of wheat,rye, barley, malt, buckwheat, sorghum (milo), millet (ragi) and rice. Inanother embodiment, the maltogenic enzyme is a beta amylase. In anotherembodiment, the maltogenic enzyme is endogenous to the grain. In anotherembodiment, the starch liquefying enzyme is an alpha amylase derivedfrom a Bacillus. In another embodiment, the starch liquefying enzyme isderived from Bacillus licheniformis or Bacillus stearothermophilus. Inanother embodiment, the transglucosidic enzyme is a transglucosidase. Inanother embodiment. The transglucosidase is derived from Aspergillus. Inanother embodiment, the Aspergillus is Aspergillus niger. The inventionalso describes a grain composition, a food additive, oral rehydrationsolution, food product and/or a flour produced according to abovedescribed method.

In another embodiment, the invention describes a method for making awheat grain composition said method comprising: (a) contacting anungelatinized wheat grain having an endogenous maltogenic beta-amylaseand a starch liquefying alpha amylase from Bacillus to produce maltose;(b) contacting said maltose with a transglucosidase, wherein said steps(a) and step (b) occur at a temperature less than wheat gelatinizingtemperature; and (c) obtaining a wheat grain composition having anenzymatically produced isomalto-oligosaccharide, wherein saidoligosaccharide is derived from said ungelatinized grain. An embodimentof the present invention is depicted in FIG. 2.

In another embodiment, the above described method can be used to make afood additive, a bakery product, oral rehydration solution and/or aflour. In another embodiment, the maltogenic enzyme is a beta amylase.In another embodiment, the maltogenic enzyme is endogenous to the grain.In another embodiment, the starch liquefying enzyme is an alpha amylasederived from a bacterial source. In one embodiment the bacterial sourceis a Bacillus sp. In another embodiment, the starch liquefying enzyme isderived from Bacillus licheniformis or Bacillus stearothermophilus. Inanother embodiment, the transglucosidic enzyme is a transglucosidase. Inanother embodiment. The transglucosidase is derived from a fungalsource. In one embodiment the fungal source is an Aspergillus sp. Inanother embodiment, the Aspergillus is Aspergillus niger. The inventionalso describes a grain composition, a food additive, oral rehydrationsolution and/or a flour produced according to above described method.The grain composition could contain greater than 1% by weight of atleast one isomalto-oligosaccharide. The at least oneisomalto-oligosaccharide can be selected from the group consisting ofisomaltose, panose, isomalto-triose. In a further embodiment of theinvention, the endogenous maltogenic enzyme is selected from betaamylase or alpha amylase. In a still further embodiment of theinvention, the solubilizing enzyme is a liquefying alpha amylase derivedfrom a Bacillus. In a still further embodiment of the invention theliquefying amylase is derived from Bacillus licheniformis or Bacillusstearothermophilus.

Substrates

The present invention includes a substrate containing a starch, forexample a grain or a tuber containing a starch that is contacted with amaltogenic enzyme and a starch liquefying enzyme to produce maltose. Theterm substrate refers to materials that can be enzymatically convertedto maltose and thus IMO's. Exemplary substrates can be at least onesubstrate selected from the group consisting of grains and tubers. Themaltose can be in the form of a maltose rich syrup or slurry.

Starch occurs in two forms, amylose, a linear chain polysaccharide, andamylopectin, a branched chain polysaccharide. Amylose contains longunbranched chains in which all the D-glucose units are linked byalpha-1,4-linkages (“α-1,4 linkages” or “1,4-α-D-glucosyl linkages”).Amylopectin is highly branched, the backbone glucosidic linkage beingα-1,4, but the branch points being α-1,6 linkages. The major componentsof starch can be enzymatically hydrolyzed in two different ways. Amylosecan be hydrolyzed by α-amylases (E.C. 3.2.1.1), e.g., α-(1-4)-glucan4-glucanohydrolase. Alpha amylases hydrolyze the α-(1,4)-linkages toyield a mixture of glucose, maltose, maltotriose and higher sugars.Amylose can also be hydrolyzed by a beta-amylase (E.C. 3.2.1.2)[alpha(1,4)-glucan maltohydrolase, 1,4-α-D-glucan maltohydrolase]. Thisenzyme cleaves away successive maltose units beginning from thenon-reducing end to yield maltose quantitatively. The alpha and betaamylases also hydrolyze amylopectin. Neither the alpha and beta amylasescan hydrolyze the alpha (1-6) linkages at the branch points ofamylopectin. The end product of exhaustive beta-amylase action onamylopectin is a large, highly branched core or beta limit dextrin. Adebranching enzyme (E.C. 3.2.1.41, e.g., pullulanases, [α-(1-6)-glucan6-glucanohydrolase, also called α-(1,6)-glucosidase]) can hydrolyze theα-(1-6) linkages at the branch points. Thus the combined action ofβ-amylase and the α 1,6-glucosidase can therefore completely degradeamylopectin to maltose and glucose, resulting in a maltose content ashigh as 60%, 65%, 79%, 75%, 80% or higher of the total sugar content.

For the purposes of this invention, the substrate containing starch canbe a grain or a tuber or mixtures thereof. The grain can be any cerealor seed containing starch. The substrate can be milled, ground orotherwise reduced in size to increase the surface area of the substratefor contacting with the respective enzymes. For example, the substratecan be wet or dry milled as desired. In one embodiment of the presentinvention, the starch is granular starch. Grains contemplated for usewithin the present invention includes any grain currently used inbaking, pasta or other uses. Exemplary grains contemplated by theinventors include, but are not limited to at least one selected from thegroup consisting of wheat (Triticum sp. Including, but not limited to T.monococcum, T. turgidum, T. spelta, and/or T. aestivum), barley (e.g.,Hordeum vulgare, and the varieties described in U.S. Pat. No. 6,492,576,Table 1), rye (Secale sp., including but not limited to S. cereale),corn (Zea sp., including, but not limited to Zea mays), buckwheat(Fagopryum sp., including, but not limited to F. esculentum), malt (forexample, germinated barley), sorghum (Sorghum sp., including, but notlimited to Sorghum bicolor) or otherwise known as milo, millet (ragi)(Panicum sp. and Setaria sp., including, but not limited to P. milaceum;and Setaria sp., including, but not limited to S. italica) and rice(Oryza sp., including, but not limited to Oryza sativa). It iscontemplated by the inventors that wild-type and transgenic plantshaving beneficial attributes, such as increased enzyme levels ofendogenous enzymes or the presence of exogenous enzymes are also usefulas starch containing substrates.

Germinated cereals, for example, malt, are used as one of the keyingredients in many food and health drink formulations because of theirhigh nutritive value, e.g., malt containing food products (TABLE A).Germination results in the synthesis and activation of endogenousmaltogenic and proteolytic enzymes. Thus germinated cereals are a goodsource of grains containing endogenous maltogenic enzymes. Malt flourand malt extract are also used as a source of digestive enzymes inbrewing and baking applications. However, germination of the barleyrenders the cereal grain components too readily digestible to play aneffective role as a prebiotic or even as a nutraceuticals, since theytend to be digested in their entirety before arrival in the lowergastrointestinal tract. Unfortunately, the beneficial effects ofprebiotic compounds are best realized in the lower gastrointestinaltract. Therefore, converting the highly digestible malto-sugars intoless digestible isomalto sugars allows for the use of the modified maltto play a role as a prebiotic, allowing the malt to arrive in the lowergastrointestinal tract and provide additional functional and healthbenefits. For example, suitable commercially available food productscontaining malt extract are provided in Table A.

TABLE A Commercial Food Products Containing Malt Extract TradenameManufacturer Location HORLICKS Glaxosmithkline Punjab, India MALTOVAGlaxosmithkline Punjab, India VIVA Glaxosmithkline Punjab, IndiaBOURNAVITA Cadbury Mumbai, India BOOST Jagjit Industries Punjab, IndiaMILO Nestle New Delhi, India

Thus the use of malt as a starch containing substrate converts some ofthe granular starch contained within the substrate to an additionalbeneficial form of the oligosaccharide, e.g., an IMO.

In addition, the substrate containing starch can be a tuber. Tuberscontemplated by the inventors include potato (Solanum sp., including,but not limited to S. tuberosum), sweet potato (Ipomoea sp., including,but not limited to Ipomoea batatas), manioc [tapioca, cassaya] (Manihotsp., including, but not limited to Manihot esculenta, Manihot aipi andManihot utilissima) and/or taro root (Colocasia sp., including, notlimited to C. esculenta or C. macrorhiza).

The amount of substrate containing starch can be an aqueous slurrycharacterized by having a concentration of 10 to 50% dissolved solids(DS). In another embodiment, the substrate containing starch ischaracterized by having a concentration of 2-90% DS. In anotherembodiment, the substrate containing starch is characterized by having aconcentration of 5-70% DS. In another embodiment, the substratecontaining starch is characterized by having a concentration of 10-60%DS. In another embodiment, the substrate containing starch ischaracterized by having a concentration of 20-40% DS. In anotherembodiment, the substrate containing starch is characterized by having aconcentration of 25-35% DS.

In another embodiment of the invention, the pH of the substratecontaining starch is between 1.00 to 9.00. In another embodiment of theinvention, the pH of the substrate containing starch is between 2.00 to8.00. In another embodiment of the invention, the pH of the substratecontaining starch is between 3.00 to 7.50. In another embodiment of theinvention, the pH of the substrate containing starch is between 4.00 to6.50. In another embodiment of the invention, the pH of the substratecontaining starch is between 4.25 to 5.75.

Enzymes

The present invention includes contacting the substrate containingstarch with a maltogenic and a starch liquefying enzyme to producemaltose. By maltogenic is meant that the enzyme is able to enzymaticallyconvert starch to maltose. Exemplary maltogenic enzymes include alphaamylases and beta amylases. As described before, amylose can behydrolyzed by αamylases (E.C. 3.2.1.1), e.g., α(1-4)-glucan4-glucanohydrolase. Alpha amylases hydrolyze the α(1,4)-linkages toyield a mixture of glucose, maltose, maltotriose and higher sugars.Amylose can also be hydrolyzed by a beta-amylase (E.C. 3.2.1.2)[alpha(1,4)-glucan maltohydrolase, 1,4-α-D-glucan maltohydrolase]. Thisenzyme cleaves away successive maltose units beginning from thenon-reducing end to yield maltose quantitatively. The alpha and betaamylases also hydrolyze amylopectin.

Alpha Amylases—

In some of the embodiments encompassed by the invention, the alphaamylase is a fungal or microbial enzyme having an E.C. number, E.C.3.2.1.1-3 and in particular E.C. 3.2.1.1. In some embodiments, the alphaamylase is a thermostable fungal alpha amylase. Suitable alpha amylasesmay be naturally occurring as well as recombinant and mutant alphaamylases. In some embodiments, the alpha amylase is derived from aBacillus species. Preferred Bacillus species include Bacillusamyloliquefaciens, B. lentus, B. licheniformis, and B.stearothermophilus. In particularly preferred embodiments, the alphaamylase is derived from a Aspergillus species. Preferred Aspergilllusspecies include Aspergillus niger and Aspergillus oryzae. Also referenceis made to strains having NCIB 11837.

Commercially available alpha amylases contemplated for use in themethods of the invention include; CLARASE L ([Aspergilus oryzae]Genencor International Inc.) and NOVAMYL ([B stearothermophilus]Novozyme Biotech.).

As understood by those in the art, the quantity of alpha amylase used inthe methods of the present invention will depend on the enzymaticactivity of the alpha amylase. In general, an amount of about 0.01 to5.0 kg of the alpha amylase is added to a metric ton (MT) of thesubstrate containing starch. Although in some embodiments the alphaamylase is added in an amount about 0.05 to 4.0 kg per MT. In otherembodiments, the alpha amylase is added in an amount of about 0.1 to 2.5kg per MT and also about 0.5 to 1.5 kg per MT. In further embodiments,other quantities are utilized. For example, generally an amount ofbetween about 0.01 to 1.5 kg of CLARASE L (Genencor International Inc.)is added to a MT of starch. In other embodiments, the enzyme is added inan amount between about 0.05 to 1.0 kg; between about 0.1 to 0.6 kg;between about 0.2 to 0.6 kg and between about 0.4 to 0.6 kg of CLARASE Lper MT of starch.

Beta Amylase

In some of the embodiments encompassed by the invention, the maltogenicenzyme is a beta amylase. While alpha amylases are maltogenic in thesense that contacting alpha amylases with a substrate containing starchwould provide maltose, the use of beta amylases are useful in that theircontact with granular starch would provide a greater amount of maltoseto the exclusion of other saccharides, e.g., glucose. In someembodiments, the beta amylase is a plant or microbial enzyme having anE.C. number, E.C. 3.2.1.2 (for example those beta amylases described inU.S. Pat. Nos. 4,970,158 and 4,647,538). In some embodiments, the betaamylase is a thermostable bacterial beta amylase. Suitable beta amylasesmay be naturally occurring as well as recombinant and mutant betaamylases. The term bacterial refers to the enzyme being derived fromBacillus sp., e.g., B. subtilis, B. licheniformis, B.stearothermophilus, B. coagulans, B. amyloliquefaciens, and/or B.lentus. Particularly preferred beta amylases are derived from Bacillusstrains B. stearothermophilus, B. amyloliquefaciens and B.licheniformis. Also reference is made to strains having NCIB 11608. Theterm plant origin refers to the enzyme being derived, extracted,isolated, expressed from a plant source, for example from barley malt,soybean, wheat or barley.

Commercially available beta amylases contemplated for use in the methodsof the invention include; OPTIMALT BBA, Spezyme DBA, and OPTIMALT ME(Genencor International Inc.). Other commercially available wheat betaamylases are also useful in the methods of the invention.

In some embodiments, the substrate containing starch, e.g., wheat, rye,barley, malt, comprises an endogenous maltogenic enzyme at sufficientlevels to produce sufficient maltose for conversion to isomaltooligosaccharides. The term “endogenous” refers to the enzyme beingpresent in the grain or tuber without having to resort to adding themaltogenic enzyme to the grain, or the grain being geneticallyengineered to provide maltogenic enzymes.

In embodiments where the substrate containing starch does not contain anendogenous maltogenic enzyme or has low endogenous levels of maltogenicenzymes, e.g., rice, millet, sorghum, and/or corn, the addition of anequivalent amount of any exogenous maltogenic enzyme is alsocontemplated by the inventors. The exogenous maltogenic enzyme can beadded, for example by genetically manipulating the host cell to expresssufficient levels of maltogenic enzyme, and/or providing a maltogenicenzyme concentrate or material from another source. The term exogenousmaltogenic enzyme refers to a maltogenic enzyme that is not presentwithin the grain. In this embodiment, a sufficient amount of maltogenicenzyme is contacted with the substrate to produce maltose.

In one embodiment, the amount of exogenous maltogenic enzyme contactedwith the substrate containing starch is between 0.050 to 5.000 Degreesof Diastatic Power (“DP°”) units/gm of maltogenic enzyme. In anotherembodiment of the invention, 0.100 to 2.000 DP° units/gm of maltogenicenzyme is contacted with the grain containing a starch. In still anotherembodiment of the invention, 0.100 to 3.000 DP° units/gm of maltogenicenzyme is contacted with the grain containing a starch.

In another embodiment, the amount of exogenous maltogenic enzymecontacted with the substrate containing starch is expressed in kilogramsof maltogenic enzyme per metric ton of substrate. In one embodiment, theamount of exogenous maltogenic enzyme contacted with the substrate isabout 0.05 kg of maltogenic enzyme per metric ton dry solids basis(“kg/mt dsb”). In another embodiment, the amount of exogenous maltogenicenzyme is about 0.1 kg of maltogenic enzyme per metric ton dry solidsbasis (“kg/mt dsb”). In other embodiments 0.2, 0.4, 0.6, 0.8. and/or 1.0kg/mt dsb provide sufficient amounts of maltogenic enzyme, e.g.,β-amylase.

In another embodiment, the amount of exogenous maltogenic enzymecontacted with the substrate containing starch is expressed in kilogramsof maltogenic enzyme per metric ton of substrate. In one embodiment, theamount of exogenous maltogenic enzyme contacted with the substrate isabout 0.05 kg of maltogenic enzyme per metric ton dissolved starch basis(“kg/mt dsb”). In another embodiment, the amount of exogenous maltogenicenzyme is about 0.1 kg of maltogenic enzyme per metric ton dissolvedstarch basis (“kg/mt dsb”). In other embodiments 0.2, 0.4, 0.6, 0.8.and/or 1.0 kg/mt dissolved starch basis provide sufficient amounts ofmaltogenic enzyme, e.g., β-amylase.

In another embodiment, the amount of maltogenic enzyme to be contactedwith the grain is in terms of maltogenic enzyme units. Assays useful todetermine the maltogenic activity include those described in theexamples and those describing β-amylase activity. The term DP° unitrefers to the amount of enzyme contained in 0.10 ml of a 5% solution ofthe sample enzyme preparation that will produce sufficient reducingsugars to reduce 5 ml of Fehling's solution when the sample is incubatedwith 100 ml of substrate for 1 hour at 20° C. (68° F.).

In another embodiment, a grain having endogenous maltogenic enzymes(barley, wheat, etc.) can be mixed with those grains needing exogenousmaltogenic enzymes. Mixtures of 30:70, 60:40, 50:50, 60:40, 70:30 grainshaving an endogenous maltogenic enzyme: grains utilizing exogenousmaltogenic enzyme sources are contemplated by the inventors, so long assufficient amounts of maltogenic enzymes are present in the mixture(endogenous or exogenous sources). Use of endogenous sources ofmaltogenic enzymes can reduce the amount of exogenous enzymes added orcontacted with the grain mixture.

Starch Liquefying Enzymes

A starch liquefying enzyme is contacted with the starch to reduce theviscosity of the liquefied or solubilized starch. In one embodiment ofthe invention the starch liquefying enzyme is an enzyme selected fromthe E.C. 3.2.1.1, e.g., alpha amylases. Exemplary alpha-amylases can bederived, isolated or extracted from a bacterial source. In oneembodiment, the bacterial source is a Bacillus. In another embodiment,the alpha-amylases derived from Bacillus include those derived from atleast one bacterial source selected from B. subtilis, B. licheniformis,B. stearothermophilus, B. coagulans, B. amyloliquefaciens, and B.lentus. Those of Bacillus licheniformis and Bacillus stearothermophilusare especially useful. Other amylases are contemplated by the inventors,for example, but not limited to those of EC 3.2.1.133 (U.S. Pat. No.6,361,809). Other amylases contemplated by the inventors include thosecharacterized by increased oxidative or thermostability, including thosemutants or genetically modified or variant amylases described in U.S.Pat. Nos. 5,763,385; 5,824,532; 5,958,739; and/or 6,008,026. Usefulalpha amylases are those derived from B. licheniformis strains NCIB8059, ATCC 6598, ATCC 6634, ATCC 8480, ATCC 9945A, ATCC 11945. Usefulalpha amylases are those derived from B. stearothermophilus strains ATCC39709. Such enzymes are identified by the trade names “SPEZYME AA” or“SPEZYME FRED”, “SPEZYME LT300”, and “SPEZYME LT75”, available fromGenencor International (Palo Also, Calif., USA). Other such enzymesinclude alpha amylases from Bacillus stearothermophilus sold under thetradename GZYME G997, GC007 and from Bacillus licheniformis sold underthe tradename GC262 SP, also available from Genencor International.

Contacting the grain containing starch with the maltogenic enzyme andthe starch liquefying enzyme produces maltose. As understood by those inthe art, the quantity of starch liquefying enzyme used in the methods ofthe present invention will depend on the enzymatic activity of thestarch liquefying enzyme. In one embodiment, 0.01 to 25 LiquefonUnits/gm of starch liquefying enzyme is contacted with the graincontaining starch. In another embodiment, 1 to 10 Liquefon Units/gm ofstarch liquefying enzyme is contacted with the grain containing astarch. One Liquefon Unit (LU) is the measure of digestion time requiredto produce a color change with iodine solution, indicating a definitestage of dextrinization of starch substrate under specified conditions.

In one embodiment, 0.1 kg of starch liquefying enzyme is added permetric ton of grain dissolved solid basis (kg/mt dsb). In otherembodiments, 0.2, 0.4, 0.4, 0.8, or 1.0 kg of starch liquefying enzymeis added per metric ton of grain (kg/mt dissolved starch basis). In oneembodiment, 0.1 kg of starch liquefying enzyme is added per metric tonof grain dissolved starch basis (kg/mt dissolved starch basis). In otherembodiments, 0.2, 0.4, 0.4, 0.8, or 1.0 kg of starch liquefying enzymeis added per metric ton of grain (kg/mt dissolved starch basis). Assaysuseful to determine the starch liquefying activity include thosedescribed in the examples herein. Exemplary assays for the determinationof α-amylase activity are also described in U.S. Pat. Nos. 5,763,385;5,824,532; 5,958,739; and/or 6,008,026 which are incorporated byreference herein.

Transglucosidic Enzyme

Contacting the maltose with a transglucosidic enzyme obtains a graincomposition having an enzymatically produced isomalto-oligosaccharide,derived from the grain containing starch. The transglucosidic enzymecatalyzes hydrolytic and transfer reactions on incubation withalpha-D-gluco-oligosaccharides to produce isomaltose, panose, kojibioseor nigerose. The presence of these sugars and thus conversion by thetransglucosidic enzyme is indicated in an increased amount of DP2disaccharides. The transglucosidic enzyme (E.C. 2.4.1.24) can betransglucosidase. Exemplary transglucosidase enzymes are identified asTRANSGLUCOSIDASE L-1000 (Genencor International, Inc.) andTRANSGLUCOSIDE L by Amano Enzymes, Inc., (Nagoya, Japan). In oneembodiment the transglucosidic enzyme is derived from a filamentousfungal source, e.g., Aspergillus sp. The transglucosidic enzyme that isderived from Aspergillus can be derived from Aspergillus niger. In oneembodiment, the Aspergillus niger strain is ATCC14916.

In this embodiment, a sufficient amount of the transglucosidic enzyme iscontacted with the substrate, e.g. the grain containing a starch toproduce maltose. As understood by those in the art, the quantity oftransglucosidic enzyme used in the methods of the present invention willdepend on the enzymatic activity of the alpha amylase. In oneembodiment, 0.01 to 25.00 transglucosidase units (“TGU”)/gm oftransglucosidase is contacted with the grain containing a starch. Inanother embodiment of the invention, 0.05 TGU to 10.00 TGU/gm oftransglucosidase is contacted with the grain containing a starch. Instill another embodiment of the invention, 0.10 to 5.00 TGU/gm of grainis contacted with the grain containing a starch. The term TGU refers tothe activity of the enzyme required to produce one micromole of panoseper minute under the conditions of the assay.

In one embodiment, 0.05 to 6.00 kg of transglucosidic enzyme is addedper metric ton of grain (kg/mt dsb). In another embodiment, 0.10 to 5.00kg of transglucosidic enzyme is added per metric ton of grain (kg/mtdsb). In another embodiment, 0.25 to 3.00 kg of transglucosidic enzymeis added per metric ton of grain (kg/mt dsb). In another embodiment,0.50 to 1.50 kg of transglucosidic enzyme is added per metric ton ofgrain (kg/mt dsb). Additional assays useful to determine thetransglucosidic activity include those described in the Examples and inShetty, J., et al (U.S. Pat. No. 4,575,487 (1986) entitled, “Method fordetermination of transglucosidase”), which are incorporated by referenceherein.

In one embodiment, 0.05 to 6.00 kg of transglucosidic enzyme is addedper metric ton of dissolved starch (kg/mt starch dsb). In anotherembodiment, 0.10 to 5.00 kg of transglucosidic enzyme is added permetric ton of grain (kg/mt starch dsb). In another embodiment, 0.25 to3.00 kg of transglucosidic enzyme is added per metric ton of grain(kg/mt starch dsb). In another embodiment, 0.50 to 1.50 kg oftransglucosidic enzyme is added per metric ton of grain (kg/mt starchdsb).

As a result of transglucosidase reactions, the malto-oligosaccharidesare converted to isomalto-oligosaccharides resulting in a new class ofpolysaccharides containing higher proportion of glucosyl residues linkedto a primary hydroxyl group of a glucose molecule from the non-reducingend. Isomalto-oligosaccharides produced by this method includeisomaltose, panose, isomalto-triose, isomalto-tetrose, isomalto-pentose,isomalto-hexose and isomalto-heptose. These sugars are receivingincreased attention as food additives because they help prevent dentalcaries (Oshima, et. al 1988, The caries inhibitory effects of gos-sugarin vitro and rat experiments. Microbial Immunol. 32.1093-1105)) andimprove human intestinal microflora acting as a growth factor(prebiotic) for bifidobacteria (Komoto, et. al 1988; Effect ofIsomalto-oligosaccharides on human fecal flora Bifidobacteria Microflora 7, 61-69).

To ascertain the production of the IMO's, assays and/or other analyticalmethods can be used to determine the amount of IMO produced. One methodfor determining the levels of IMO produced includes high performanceliquid chromatography (HPLC). For example, analysis of the mixture canprovide an indication of the levels of the various sugars produced bythe process. A useful rating is the degree of polymerization (DP) of themixture. The term degree of polymerization is a measure of the relativeamounts of the number of glucose residues in the molecule. For example,glucose (one glucosyl unit, the lowest level of polymerization) isusually found as DP1. Isomalto-oligosaccharides are usually found in DP2(two glucosyl units). In one embodiment, the grain composition containsgreater than at least 1%, at least 5%, at least 25%, at least 40%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70% byweight of at least one isomalto-oligosaccharide. In one embodiment theat least one isomalto-oligosaccharide is selected from the group ofisomaltose, panose and/or isomalto-triose. In one embodiment, the amountof isomalto-oligosaccharides produced in the grain composition isbetween 1% and 99% of the grain composition. In one embodiment, theamount of isomalto-oligosaccharides produced in the grain composition isbetween 1% and 90% of the grain composition. In one embodiment, theamount of isomalto-oligosaccharides produced in the grain composition isbetween 1% and 80% of the grain composition. In one embodiment, theamount of isomalto-oligosaccharides produced in the grain composition isbetween 1% and 70% of the grain composition. In one embodiment of thepresent invention, the total sugar present in the grain compositionafter the above described procedure includes a level of maltose in thetotal sugar content of greater than 50%, greater than 60%, greater than70%, or greater than 80%. Levels of maltose greater than 50% includeranges from 50% to 85%, from 55% to 80%, and/or from 60% to 75%. Theterm RBS ratio refers to the ratio of maltose (DP2) present in the grainas compared to the level of maltotriose (DP3) present in the resultantgrain composition. A higher RBS value indicates a higher amount ofmaltose present and thus a more complete conversion of the starch tomaltose as opposed to the presence of other less desirable end-products,e.g., maltotriose. In one embodiment, the RBS ratio is greater than 2.0.In one embodiments, the RBS ratio is greater than 3.0, greater than 4.0.Exemplary ranges include an RBS ratio of 2.0 to 50.0, 2.0 to 30.0 and/or2.0 to 10.0. Various RBS ratios are described in the examples. It isnoted that the hydrolysis of liquefied starch by commercialbeta-amylases (barley or wheat) generally produces a maltose contentbetween 55% and 65%. For maltose content greater than 50% usingliquefied starch, the addition of debranching enzyme and/or a very lowstarting DE of the liquefied starch was previously required. Optionally,the addition of a debranching enzyme can be used to increase theproduction of maltose. The term debranching enzyme refers to enzymesthat catalyze the hydrolysis of α-1,6-linkages. An enzyme of the classE.C. 3.2.1.41 is useful in this regard. An exemplary enzyme of thisclass is a pullanase, also known as α-dextrin endo-1,6-α glucosidase,limit dextrinase, debranching enzyme, amylopectin 1,6-glucanohydrolase.

Contacting the grain containing starch with a maltogenic enzyme toproduce the maltose and the contacting of the maltose with atransglucosidic enzyme occurs at a temperature less than thegelatinization temperature of the starch of the grain used. The enzymesare contacted or incubated with the respective enzymes for an incubationtime of at least 12 hours, at least 18 hours, at least 24 hours, atleast 30 hours and/or at least 36 hours. The period of at least a notedtime refers to a period of 12-80 hours, at least 18-60 hours and/or atleast 24-48 hours. The term incubation time refers to the period of timefor the conversion of maltose or the substrate to IMO's. Thetransglucosidic enzyme can be contacted or added separately orconcurrently with the substrate, e.g. grain containing starch, themaltogenic enzyme, e.g., the alpha amylase or beta amylase, and/or theliquefying enzyme, e.g., the alpha amylase. In one embodiment, thetransglucosidic enzyme is added concurrently with the liquefying enzyme.Thus in one embodiment, steps (a) and (b) are performed concurrently. Inanother embodiment, the steps (a) and (b) are performed sequentially orseparately. In another embodiment, the step (a) is performed before step(b). The term gelatinization temperature refers to the temperature atwhich the starch contained within the grain changes phases orgelatinizes. While the specific temperature varies from grain to grain,temperatures sufficient to effect the gelatinization of starch includethose greater than 45° C., greater than 50° C., greater than 60° C.,greater than 70° C., greater than 80° C., and/or greater than 90° C.Exemplary temperatures greater than the indicated gelatinizationtemperatures include 45° C. to 120° C., 50° C. to 110° C., 500 to 100°C. In one embodiment, e.g. wheat, the gelatinization temperature is atemperature the grain is kept below, e.g. a temperature selected frombelow 50° C. to 70° C., in another embodiment below 55° C. to 65° C.,and in another embodiment, below 60° C. For example, gelatinizationtemperatures have been described for corn, potato, wheat, tapioca, waxymaize, sorghum, rice sago, arrowroot amylomaize and/or sweet potato asshown in Table 1 (Beynum, G. M. A and Roels, J. A., Starch ConversionTechnology (Marcel Dekker, Inc., New York, N.Y. (1985), pp. 15-45):

TABLE 1 Kofler gelatinization Brabender temperature pasting Starch range(° C.) temperature (° C.) Corn 62-67-72 75-80 Potato 58-63-68 60-65Wheat 58-61-64 80-85 Tapioca 59-64-69 65-70 Waxy Maize 63-68-72 65-70Sorghum 68-74-78 75-80 Rice 68-74-78 70-75 Sago 60-66-72 65-70 Arrowroot62-66-70 Amylomaize 67-80-92 90-95 Sweet Potato 58-65-72 65-70

In another embodiment of the present invention, the slurry, after theincubation time, can be subjected to a flash heat period sufficient tohalt further enzymatic activity, but not gelatinize or liquefy theslurry. For example, the slurry can be heated to a temperature of 80°,85°, 90° 95° or 100° C. for a period of 5-60 minutes, 10.0 to 40.0minutes or 30.0 minutes.

Another embodiment of the present invention further includes the step ofseparating the slurry into insolubles and solubles. The separating stepcan be by any chromatographic method known in the arts, for example, butnot limited to HPLC, size exclusion and/or charge chromatography.Filtering can be used to separate the insolubles from the solubles. Theinsolubles or entire slurry can be subjected to the drying stepsdescribed later in this application. In another embodiment, the solublesresulting from the separating step can be concentrated by evaporation,for example by roto-evaporation, tray drying, etc. The evaporatedconcentrate can be subjected to carbon treatment (filtered throughcarbon granules) and/or further chromatographic treatment to provide anisolated IMO liquid concentrate. The isolated IMO concentrate can havean IMO concentration of greater than 75%, greater than 85%, greater than90%, greater than 95%, greater than 97%, and/or greater than 99%.

Another embodiment of the present invention is the use or incorporationof such syrup (the isomalto-oligosaccharides enzymatically derived fromthe substrate having ungelatinized starch) in oral rehydrationsolutions. The amount of the isomalto-saccharides can be in the amountsor formulations as described as U.S. Pat. Nos. 4,981,687; 5,096,894;and/or 5,733,579.

Another embodiment of the present invention is the drying of theaforementioned isomalto-oligosaccharide substrate, grain or tubercomposition to produce a powder including the grain composition.Typically, this drying step is accelerated by heating. The graincomposition can be dried to a desired moisture level by using a suitabledrying method, for example, but not limited to a spray dryer, traydryer, tumble dryer, drum dryer or cabinet dryer. Other dryingmethodologies can be used, for example spray drying, evaporative dryingunder reduced pressure.

By drying the grain composition, slurry, separated insolubles, and/orseparated solubles, a flour or other dried powder is obtained therefrom.The resulting powder or flour can be incorporated into compositions inwhich the presence of isomalto-oligosaccharides is desired, for examplein food stuffs (breakfast cereals, pastas), food additives and bakedgoods. The term food additive refers to the use of theisomalto-oligosaccharides as a sprinkle on material, as an ingredientfor use in the manufacture of other foods, and/or a topical ingredientadded to the food.

In another embodiment, the dried powder can be incorporated into foodsupplements. The incorporation of the dried powder into a foodsupplement can be provided in any acceptable supplement or form. Thedietary supplements can be formulated for oral administration in amatrix as, for example but not limited to, drug powders, crystals,granules, small particles (which include particles sized on the order ofmicrometers, such as microspheres and microcapsules), particles (whichinclude particles sized on the order of millimeters), beads, microbeads,pellets, pills, microtablets, compressed tablets or tablet triturates,molded tablets or tablet triturates, and in capsules, which are eitherhard or soft and contain the composition as a powder, particle, bead,solution or suspension. The dietary supplement can also be formulatedfor oral administration as a solution or suspension in an aqueousliquid, as a liquid incorporated into a gel capsule or as any otherconvenient form for administration or for rectal administration, as asuppository, enema or other convenient form. Theisomalto-oligosaccharide composition can also be provided as acontrolled release system.

The dietary supplement formulation can also include any type ofacceptable excipients, additives or vehicles. For example, but not byway of limitation, diluents or fillers, such as dextrates, dicalciumphosphate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodiumchloride, dry starch, sorbitol, sucrose, inositol, powdered sugar,bentonitc, microcrystalline cellulose, or hydroxypropyl methylcellulosemay be added to isomalto-oligosaccharide composition to increase thebulk of the composition. Also, binders, such as, but not limited to,starch, gelatin, sucrose, glucose, dextrose, molasses, lactose, acaciagum, sodium alginate, extract of Irish moss, panwar gum, ghatti gum,mucilage of isapgol husks, carboxymethylcellulose, methylcellulose,polyvinylpyrrolidonc, Veegurn and starch arabogalactan, polyethyleneglycol, ethylcellulose, glycerylmonostearate and waxes, may be added tothe formulation to increase its cohesive qualities.

Additionally, lubricants, such as, but not limited to, glycerylmonostearate, talc, magnesium15 stearate, calcium stearate, stearicacid, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, carbowax, sodium laurylsulfate, and magnesium lauryl sulfate may be added to the formulation.Also, glidants, such as but not limited to, colloidal silicon dioxide,magnesium silicate or talc may be added to improve the flowcharacteristics of a powdered formulation. Finally, disintegrants, forexample, but not limited to, starches, clays, celluloses, algins, gums,crosslinked polymers (e.g., croscarmelose, crospovidone, and sodiumstarch glycolate), Veegum, methylcellulose, agar, benton:ite, celluloseand wood products, natural sponge, cation-exchange resins, alginic acid,guar gum, citrus pulp, carboxymethylcellulose, or sodium lauryl sulfatewith starch may also be added to facilitate disintegration of theformulation in the stomach or intestine.

Another embodiment of the present invention is the use of the novelsubstrate, tuber or grain composition described herein in the productionof flour for use in various baked goods. The term baked goods refers toleavened and unleavened goods. The term leavened refers to baked goodsusing yeast in the baking process. Whereas the term unleavened meansbaked goods not using yeast in the baking process. Exemplary goodsinclude bread, cookies, cakes, pies, biscuits, naan, bagels, pasta,crackers, rolls, donuts, pitas and pastries. Exemplary unlevened goodsinclude matzoh, chapathi, breakfast cereals and tortillas. Anotherembodiment of the present invention is the use of the novel graincompositions in pasta, for example, noodles (penne, spaghetti, lasagna,udon, etc.). Another embodiment of the present invention is a substrate,tuber or grain composition made according to the above-described method.Another embodiment of the present invention is a flour comprising thesubstrate, tuber or grain composition made according to the abovedescribed method. Another embodiment of the present invention is an oralrehydration solution comprising the isomalto-oligosaccharide describedabove. Flour comprising the substrate, tuber or grain composition can bemade according to the above described method. Another embodiment of thepresent invention is a substrate, tuber or grain composition madeaccording to the above-described method.

Another embodiment of the present invention is a substrate, tuber orgrain composition made according to the above-described method. Anotherembodiment of the present invention is the use of the novel graincompositions in fermentive/beer worts or substrates. For example, thenovel grain composition can be used as described in beer fermentation asdescribed in International Publication No WO 02/20712 A1, which isincorporated by reference herein. The novel grain compositions can alsobe incorporated in beer adjuncts.

The isomalto-oligosaccharide containing substrate can also be subjectedto an additional step of recovering the maltose by extraction andisolation of the generated maltose, for example as a maltose syrup. Thesyrup can be extracted and/or isolated from the grain composition bymethods familiar in the art, for example in U.S. Pat. Nos. 3,922,196 and4,113,509, which are incorporated by reference herein.

Another route to enhance the sweetness or the isomalto-oligosaccharidecontent, is to treat the produced isomalto-oligosaccharide syrup with ahydrolase (in soluble or immobilized form) which hydrolysespreferentially or even exclusively malto-oligosaccharides, and has onlya small or even no affinity for isomalto-oligosaccharides. Examples ofsuch an enzyme is glucoamylase from A. niger or other sources likeAspergillus sp. or Rhizopus sp. which preferentially hydrolysesmalto-oligosaccharides (Manjunath P., Shenoy B. C., Raghavendra Rao M.R., Journal of Applied Biochemistry, 5(1983),235-260; Meagher M. M., etal., Biotechnology and Bioengineering, 34(1989), 681-693; Pazur J. H.,Kleppe K., The Journal of Biological Chemistry, 237(4)(1962),1002-1006;Hiromi K., Nitta Y., et al., Biochimica et Biophysica Acta,302(1973),362-37).

Also an enzyme like the alpha-D-glucopyranosidase from Bacillusstearothermophilus can be applied. This enzyme is not capable ofhydrolysing isomalto-oligosaccharides and will only degrade themalto-oligosaccharides present in the isomalto-oligosaccharide richsyrup (Suzuki Y., Shinji M., Nobuyuki E., Biochimica et Biophysica Acta,787(1984),281-289). Also other alpha-D-glucosidases which are calledmaltases can be used. The maltase from yeast for example will onlyhydrolyse maltose and to a lesser extent maltotriose (Kelly C. T.,Fogarty W. M., Process Biochemistry, May/June(1983),6-12).

After the hydrolysis of the malto-oligosaccharides to glucose, the syrupcan be enriched in isomalto-oligosaccharides by a chromatographictechnique or by nano- or ultra-filtration.

The following examples serve to illustrate the main embodiments of thisinvention.

EXAMPLES

The following specific examples further illustrate the compositions andthe methods of the invention. It is to be understood that these examplesare for illustrative purposes only and can be applied to any othersuitable materials rich in starch and containing endogenous maltoseproducing enzyme, for example, wheat, rice, barley, malt, potato, sweetpotato, etc.

Enzyme Activity Determination

The transglucosidase activity is measured by the method of Shetty, J.,et al, 1986 (U.S. Pat. No. 4,575,487).

The beta amylase activity was measured by a 30-minute hydrolysis of astarch substrate at pH 4.6 and 20° C. The reducing sugar groups producedon hydrolysis are measured in titrimetric procedure using alkalineferricyanide. One unit of diastase activity, expressed as degrees DPrefers to the amount of enzyme, contained in 0.1 ml of 5% solution ofthe sample enzyme preparation that will produce sufficient reducingsugars to reduce 5 mL of Fehlings' solution when the sample is incubatedwith 100 mL of the substrate for 1 hour at 20 C.

The alpha amylase activity was developed based on an end-point assay kitsupplied by Megazyme (Aust.) Pty. Ltd. A vial of substrate(p-nitrophenyl maltoheptaoside, BPNPG7) was dissolved in 10 ml ofsterile water followed by a 1:4 dilution in assay buffer (50 mM maleatebuffer, pH 6.7, 5 mM calcium chloride, 0.002% Tween20). Assays wereperformed by adding 10 μl of amylase to 790 μl of the substrate in acuvette at 25° C. Rates of hydrolysis were measured as the rate ofchange of absorbance at 410 nm, after a delay of 75 seconds. The assaywas linear up to rates of 0.2 absorption units/min.

α-Amylase protein concentration was measured using the standard Bio-RadAssay (Bio-Rad Laboratories) based on the method of Bradford, Anal.Biochem., Vol. 72, p. 248 (1976) using bovine serum albumin standards.

Substrates:

The wheat flour used as substrates in all examples, was purchased fromretail commercial stores. Other tuber or grain substrates, e.g., riceand barley used as substrates may be purchased from commercial sources(Huai An Liujun Food processing company, Jiangshu province, China).

Oligosaccharide Analysis

The composition of the reaction products of oligosaccharides wasmeasured by HPLC (Agilent 1010, Palo Alto, Calif., USA) equipped with aHPLC column (Rezex 8 u8% H, Monosaccharides), maintained at 60° C.fitted with a refractive index (RI) detector (ERC-7515A, RI Detectorfrom The Anspec Company, Inc.). Dilute sulfuric acid (0.01 N) was usedas the mobile phase at a flow rate of 0.6 ml per minute. Twentymicroliter of 4.0% solution was injected on to the column. The columnseparates based on the molecular weight of the saccharides. For examplea designation of DP1 is a monosaccharide, such as glucose; a designationof DP2 is a disaccharide, such as maltose; a designation of DP3 is adisaccharide, such as maltotriose and the designation DP4⁺ is anoligosaccharide having a degree of polymerization (DP) of 4 or greater.The term Higher sugar (“Hr. Sugar”) refers to sugars having DP greaterthan 3.

For iso-saccharides or branched sugars, the reaction products weremeasured by HPLC (Agilent 1010, Palo Alto, Calif., USA) equipped with aHPLC column (Shodex Rspak Oligosaccharide Column #DC-613), maintained at50° C. fitted with a refractive index (RI) detector (ERC-7515A, RIDetector from The Anspec Company, Inc.). 70 (Acetonitrile):25(methanol):5 Water was used as the mobile phase at a flow rate of 2.5 mlper minute. Twenty microliter of 4.0% solution was injected on to thecolumn. The column separates based on the molecular weight of thesaccharides. The standard sugars, glucose, maltose, maltotriose,isomaltose, panose and isomalto-triose (Sigma Chemicals, St. Louis, Mo.,USA) were used to calibrate the column.

Example 1

Maltose production from wheat flour by alpha amylase from Bacilluslicheniformis (an alpha amylase sold under the tradename GC262SP byGenencor International, Palo Alto, Calif.) and Bacillusstearothermophilus (an alpha amylase sold under the tradename GC007 byGenencor International, Palo Alto, Calif.) were compared. One hundredfifty grams of wheat flour from commercial retail sources was suspendedin 450 ml of deionized water. The suspension was stirred for 15 minutesat room temperature for uniform mixing (pH 5.5). The pH was adjustedwith 6.0 N sulphuric acid (H₂SO₄). The resultant suspension was kept ina water bath maintained at 60° C. and stirred for uniform mixing beforethe enzymes were added. About 6,000 LU/g of amylase from Bacillusstearothermophilus (0.6 kg of GC007 [from Genencor International.Inc.]/Metric ton (Mt) starch dsb) and 15,100 LU/g of amylase fromBacillus licheniformis (0.6 kg GC262 SP from [Genencor International.Inc.]/Mt. starch dsb) were added separately and incubated with constantstirring at 60° C. Samples were withdrawn at different predeterminedintervals of time and analyzed for total sugar composition usinghigh-pressure liquid chromatography (HPLC). Two ml of sample was takenfrom each container at a predetermined time interval using a plasticpipette and transferred to a centrifuge tube. The sample was centrifugedat 8000 prm for 3 minutes. The supernatent was withdrawn from thecentrifuge tube and a few drops were placed into a sample well of aLecia AR200 (Leica Microsystems, Inc., Buffalo, N.Y., USA) digital handheld refractometer and recorded (Table 2). The Brix (as a measure of thedissolved sugars) of the solution was determined (Table 2).

TABLE 2 Comparison of liquefying alpha amylases on the production ofmaltose during incubation of wheat flour at pH 5.5, 60° C. IncubationTime, After 1 hour at 60° heating at Sugar & Percent Sugar 80° C., 30Enzyme & Dosage BRIX 2 hours 4 hours 6 hours minutes No added Alpha DP13.86 3.70 3.74 Gelati- Amylase DP2 54.87 56.24 56.78 nized DP3 2.83 3.443.96 Hr. Sugar 38.44 36.62 35.52 BRIX 11.00 12.70 14.10 B. DP1 2.67 2.893.23 3.17 Stearothermophilus DP2 65.10 67.44 69.08 65.83 [GC007] DP310.62 12.18 13.07 13.42 0.6 kg/MT starch Hr. Sugar 21.61 17.49 14.6217.58 .dsb BRIX 18.50 19.40 20.00 22.70 B. licheniformis DP1 2.86 3.593.96 4.03 [GC262SP] DP2 62.43 64.36 65.88 58.14 0.6 kg/MT starch DP313.19 14.82 15.93 16.69 .dsb Hr. Sugar 21.52 17.23 14.23 21.14 BRIX17.40 18.40 19.10 22.60

The results in Table 2 showed that wheat flour incubated with alphaamylase from Bacillus stearothermophilus gave a higher maltose contentcompared to the maltose content from the incubation with alpha amylasefrom Bacillus licheniformis. Incubation of wheat flour with alphaamylases resulted in a significant increase in the dissolved solids dueto the hydrolysis of the granular starch compared to incubation of wheatflour without alpha amylase addition. It is interesting to note herethat the reaction product of alpha amylase from Bacillusstearothermophilus resulted in a higher ratio of maltose to glucose andmaltose to maltotriose compared to alpha amylase from Bacilluslicheniformis. These results indicate that the alpha amylase fromBacillus stearothermophilus is an especially useful enzyme for producingvery high maltose syrup.

Example 2

Effect of Bacillus stearothermophilus alpha amylase (alpha amylase soldunder the tradename GC 007 by Genencor International, Palo Alto, Calif.)concentration on the maltose production during incubation with wheatflour. The experimental conditions were identical as explained inExample 1. In addition, Bacillus stearothermophilus (6,000 Units/g) wasadded at 0.1 Kg, 0.2 Kg and 0.6 Kg/MT of starch dsb. The results aresummarized in table 3.

TABLE 3 Effect of Alpha Amylase [GC007] Concentration on Maltose Yieldduring incubation of wheat flour, pH 5.5, 60° C. Incubation Time, 1 hourat 60° GC007 DP & Percent Sugar concentration BRIX 2 hours 4 hours 6hours 24 hours 0.1 kg/MT DP1 1.87 2.10 2.27 3.70 starch.dsb DP2 65.5664.25 65.63 70.22 DP3 5.92 7.15 8.03 11.68 Hr. Sugar 29.65 26.50 24.0714.40 BRIX 17.80 19.20 19.70 21.80 0.2 kg/MT DP1 1.71 2.13 2.37 3.90starch.dsb DP2 62.47 64.34 65.44 69.30 DP3 6.75 7.78 8.58 12.27 Hr.Sugar 29.07 25.75 23.61 14.53 BRIX 18.00 19.20 19.80 21.80 0.6 kg/MTstarch DP1 2.67 2.89 3.23 3.17 .dsb DP2 65.10 67.44 69.08 65.83 DP310.62 12.18 13.03 13.42 Hr. Sugar 21.61 17.49 14.62 17.58 BRIX 18.5019.40 20.00 22.70

No significant effect of different levels of alpha amylase during theincubation of wheat flour was noticed either on the maltose content oron the dissolved solids. So, for further optimization studies, 0.1 Kg ofGC007/MT. starch, dsb was used.

Example 3

One hundred fifty grams of wheat flour was suspended in 450 ml ofdeionized water and the pH was adjusted to pH 5.00, 4.50 and 4.00 using6.0 N H₂SO₄. The slurry was stirred well for uniform mixing and the pHwas adjusted until the specified pH was stabilized. GC007 was added at0.1 Kgs/MT, starch dsb to each of the trial and incubated at 60° C. Thesamples were withdrawn at different predetermined intervals of time andthe composition of the sugar and brix were measured as described inExample 1. (Table 4).

TABLE 4 Effect of pH on the maltose yield during incubation of wheatflour with GC007 Incubation Time, 1 hour at 60° DP & Percent Sugar PHBRIX 2 hours 4 hours 6 hours 24 hours 5.0 DP1 2.59 3.22 3.77 5.17 DP263.87 66.89 67.41 65.77 DP3 10.90 13.23 14.25 16.87 Hr. Sugar 22.6416.66 14.57 12.19 BRIX 17.70 18.80 19.20 20.90 4.5 DP1 2.29 2.80 3.314.37 DP2 63.86 67.44 68.03 65.97 DP3 10.93 13.30 14.44 17.56 Hr. Sugar22.92 16.46 14.22 19.10 BRIX 17.60 18.60 19.10 20.70 4.0 DP1 1.71 1.792.11 3.17 DP2 58.19 60.23 61.28 62.95 DP3 11.16 13.47 14.87 18.67 Hr.Sugar 28.93 24.51 21.74 15.21 BRIX 17.60 18.60 19.00 19.50

The maltose content increased with decreasing pH of the incubation ofthe wheat flour from pH 5.5 and reached maximum of about 68% at pH 4.5followed by a decrease at pH 4.0. This is an unexpected result showingthe production of maltose content greater than 60% without the additionof a debranching enzyme during the hydrolysis of starch with plant betaamylases. The hydrolysis of liquefied starch by commercial Beta amylases(barley or wheat) generally produces maltose content between 55% and60%. For maltose content greater than 60% using liquefied starch, theaddition of debranching enzyme and or a very low starting DE of theliquefied starch are required. It is also important to note here thatthe process described in this invention allows maltose manufacturers toprocess at pH 4.5 and 60° C. that reduces the high risk of microbialcontamination of the current process.

Example 4

One hundred fifty grams of wheat flour was suspended in 450 ml ofdeionized water and the pH of the slurry was adjusted to pH 4.5. Theslurry was stirred well for uniform mixing and the pH was adjusted with6.0 N H₂SO₄ until the pH was stabilized. The resultant suspension waskept in a water bath maintained at 60° C. and stirred for uniform mixingbefore the enzymes were added. A starch liquefying enzyme, e.g., aBacillus stearothermophilus alpha amylase sold under the tradename “GC007” (Genencor International, Inc.) was added at 0.1 Kg/MT, dsb. Adebranching enzyme, a pullulanase sold under the tradename OPTIMAXL-1000 (Genencor International, Inc.) was then added at 0.25 Kg, 0.5 Kgand 1.0 Kg/M T dsb and incubated at 60° C. The samples were withdrawn atpredetermined different intervals of time (2, 4, 6 and 24 hours) and thecomposition of the sugar and brix were measured as described inExample 1. The results were recorded (Table 5).

TABLE 5 Effect of debranching enzyme (OPTIMAX L-1000) on the maltoseyield during incubation of wheat Flour with GC007 (0.1 Kg/MT, dsb) at pH4.5, 60° C. Incubation Time, 1 hour at 60° OPTIMAX DP & Percent Sugarconcentration BRIX 2 hours 4 hours 6 hours 24 hours 0.0 DP1 1.21 1.381.47 1.99 DP2 62.77 64.69 66.24 67.16 DP3 6.29 7.32 8.64 10.97 Hr. Sugar29.73 26.61 23.65 19.90 BRIX 17.5 18.6 19.4 20.40 0.25 Kg/MT dsb DP11.28 1.33 1.90 1.94 DP2 66.61 69.97 72.01 73.64 DP3 8.18 10.03 10.6813.58 Hr. Sugar 23.93 18.67 15.41 10.86 BRIX 17.70 18.60 19.40 20.800.50 Kg/MT dsb DP1 1.41 1.41 1.64 2.01 DP2 70.50 73.35 74.46 74.32 DP39.62 11.26 11.94 14.31 Hr. Sugar 18.47 13.98 11.96 9.36 BRIX 17.70 18.7019.80 20.80 1.00 Kg/MT dsb DP1 1.47 1.56 1.58 2.02 DP2 72.90 75.54 76.0875.21 DP3 10.05 11.35 11.95 13.53 Hr. Sugar 15.48 11.55 10.39 9.24 BRIX17.60 18.80 19.70 21.70

Maltogenic enzymes (such as beta amylases) or starch liquefying alphaamylases (such as GC007) can not hydrolyze the alpha 1-6 glucosidiclinkages, the branch point in the amylopectin of the starch substrate.So it is a common practice to add debranching enzyme, pullulanase(OPTIMAX L-1000 from Genencor International Inc) for producing maltosegreater than 65% during the incubation of starch substrate with betaamylase. The effect of OPTIMAX L-1000 concentration during theincubation of wheat flour with GC007 was studied and the results wereshown in Table 5. OPTIMAX L-1000 addition resulted in a significantlyhigher level (>75%) of maltose (DP2) compared to the control.

Example 5

It is generally the common practice in the industry to use high maltosesyrup produced by an enzymatic process using a high temperature (>90°C.) enzyme liquefied starch substrate followed by treating with enzymeglucosyltransferase for producing isomalto-oligosaccharides syrups. Thisexample illustrates the process of converting the granular starch in thewheat flour into isomalto-oligosaccharides in a single step. In thisexample, 275 grams of wheat flour was placed in a flask and 688 ml ofdeionized water was added. It was then stirred for 15 minutes foruniform mixing and the pH was then adjusted to pH 4.5 using 6.0 N H₂SO₄.The resultant suspension was kept in a water bath maintained at 60 C andstirred for uniform mixing before the enzymes were added. A starchliquefying enzyme, e.g., Bacillus stearothermophilus alpha amylase([GC007 supplied by Genencor International] (0.1 Kgs/MT dsb) and adebranching enzyme, e.g., a pullulanase (OPTIMAX L-1000 supplied byGenencor International) (0.5 kgs/MT dsb) were added. The suspension wasthen divided into two equal parts. To one of the parts, an Aspergillusniger transglucosidase sold under the tradename “TRANSGLUCOSIDASE L-500”(Genencor International) was added at 1.0 Kg/MT dsb and kept in a waterbath maintained at 60° C. (Sample 1). The other part was incubated firstfor four hours at 60°, followed by the addition of Aspergillus nigertransglucosidase sold under the tradename “TRANSGLUCOSIDASE L-500”(Genencor International) at 1.0 Kg/MT dsb and maintenance in a waterbath maintained at 60° C. (Sample 2).

The results shown in Table 6 indicate that conversion of the substrateto IMO's occurs with or without preincubation of the substrate (wheatflour) prior to the addition of the transglucosidase.

TABLE 6 Formation of isomalto-saccharides during the incubation of wheatflour with GC007 and OPTIMAX L-1000 at pH 4.5, 60° C., byTRANSGLUCOSIDASE L-500 Reaction Branch IMO Sample Time Glucose MaltoseIsomaltose Maltotriose Panose Isomaltotriose G3+ No. 1 17 14.74 30.272.73 6.82 23.05 7.13 14.65 47.56 2 17 13.92 33.17 2.32 7.70 23.69 4.8814.32 45.21 1 48 16.90 20.04 7.98 10.29 22.35 3.95 17.95 52.23 2 4814.96 23.59 6.85 12.17 16.65 5.57 19.66 48.73 1 72 16.78 21.21 5.78 8.5122.83 4.17 20.24 53.02 2 72 15.24 25.44 4.99 8.88 18.48 7.52 19.03 50.01IMO No. is calculated as the sum of isomaltose, panose, isomaltotrioseand branched sugars greater than DP3

Incubation of modified wheat flour containing high content of maltosewith Transglucosidase produced isomalto-oligosaccharides identical tothe composition produced by the conventional process. The process issimple, economical and can be easily scaled to commercial production

Example 6

It is common knowledge that cereals like wheat, barley and rye containhigh levels of beta-amylase. Incubation of these cereals at 55°-60° C.,pH 5.5 generally results in syrups containing greater than 50% maltose.A 28% slurry of wheat, barley and rye flour, respectively, was eachprepared by adding 280 grams of the respective flour to 720 gm ofdeionized water. To each of these preparations, a Bacillusstearothermophilus alpha amylase (e.g., a Bacillus stearothermophilusalpha amylase sold under the trademark “GC007” by GenencorInternational) was added at 0.2 kg/MT of the flour. The pH was thenadjusted to pH 5.5 using 6.0 N H₂SO₄ and incubated at 60° C. for 4.5hours. The pH of the incubated samples was then adjusted to pH 4.5 using6 N H₂SO₄ and 1.25 kg of transglulcosidase (e.g., a transglucosidasesold under the tradename TRANSGLUCOSIDASE L-500 by GenencorInternational)/MT of the flour was added. The slurries were thenincubated at 60° C. water bath for 48 hours. The samples were thencentrifuged and analyzed for IMO composition (Table 7) as set forth inExample 1.

TABLE 7 Soluble Carbohydrate Composition of Wheat, Barley and Rye AfterIncubating with GC007 and “TRANSGLUCOSIDASE L-500” Soluble CarbohydrateComposition % Grains/ Time Branch IMO Cereals Treatment (hours) GlucoseMaltose Isomaltose Maltotriose Panose Isomaltotriose G3+ No. Wheat TgL-500* 48 21.92 19.37 9.97 3.67 26.52 3.12 14.85 54.46 Barley Tg L-500*48 25.43 6.68 16.50 7.72 12.82 0.30 30.54 60.16 Rye Tg L-500* 48 22.1810.39 11.15 3.21 22.93 0.01 30.14 64.22 *Tg L-500 means“TRANSGLUCOSIDASE L-500”

Example 7

In an experiment, 140 grams of malt (Cargill Malt/Schreier-MaltingCompany, Wisconsin, USA) was mixed with 360 grams of distilled water.The slurry was stirred for 15.0 minutes at room temperature for uniformmixing and pH was then adjusted to pH 4.5 using dilute acetic acid.After stabilization of the pH, the slurry was kept in a water bathmaintained at 60° C. Incubation was continued for 30.0 minutes withconstant stirring and a 2 ml sample was withdrawn for Brix and HPLCanalysis (0, time). Transglucosidase L-500 was added at 1.5 kg/MT maltand incubated at 60° C. Samples were withdrawn at differentpredetermined intervals of time during incubation, e.g., 2, 4, 6, 12,and 24 hours, to ascertain Brix and IMO composition (Table 8) asdescribed in Example 1.

TABLE 8 Soluble Carbohydrate Composition of Malt After Incubating withTRANSGLUCOSIDASE L-500 Soluble Carbohydrate Composition % Grains/ TimeBranch IMO Cereals (hours) Brix Glucose Maltose Isomaltose MaltotriosePanose Isomaltotriose G3+ No. Malt 0 15.70 16.42 33.87 — 14.04 — — 35.66— Malt 2 16.50 19.59 21.91 0.75 7.70 9.38 2.87 31.40 44.40 Malt 4 16.7021.82 19.70 0.90 6.16 12.35 1.42 30.94 45.62 Malt 6 16.90 24.43 18.291.00 4.63 11.50 2.51 30.73 45.84 Malt 12 17.00 28.37 15.74 3.93 4.328.36 2.68 29.10 43.98 Malt 24 17.00 33.24 14.94 5.96 2.34 5.50 3.3426.15 40.95

The results in Table 8 showed that the commercial malt extract can beused as a suitable substrate for producing malt extract containingisomalto-oligosaccharides. The reaction time and the composition of IMOsugars of the malt extract could be adjusted by controlling the enzymedosage. The addition of maltogenic enzymes can increase IMO content ofthe resulting composition.

Example 8 Sorghum, Millet and Rice (Exogenous Maltogenic Enzymes)

In another experiment, 280 grams of sorghum, millet and rice flour wereeach taken separately and mixed separately with 720 grams of deionizedwater.

The pH of the suspension was adjusted to pH 5.5 and a Bacillusstearotherphilus alpha amylase sold under the trademark “GC007”(Genencor International) was added at 0.5 kg/mt of the flour. Afteruniform mixing, the suspension was kept in a water bath maintained at75° C. The reaction mixture was continuously stirred during incubationfor 6 hours. The temperature was then reduced to 60° C. and abeta-amylase (sold under the tradename OPTIMALT BBA by GenencorInternational) was added at 1.0 kg/mt of the flour. The incubation wascontinued for additional 10-15 hours (a sample was taken for Brix andHPLC). After the specified time, the pH was reduced to pH 4.5 by 6NH₂SO₄ and an Aspergillus niger transglucosidase (sold under thetradename “TRANSGLUCOSIDASE L-500” by Genencor International) was addedat 1.0 kg/mt flour. Samples were taken at 24 and 48 hrs. for analysis(Table 9).

TABLE 9 Soluble Carbohydrate Composition of Sorghum, Millet and RiceAfter “TRANSGLUCOSIDASE L-500” Treatment Soluble CarbohydrateComposition % Grains/ Time Branch IMO Cereals Treatment (hours) BrixGlucose Maltose Isomaltose Maltotriose Panose Isomaltotriose G3+ No.Sorghum Tg L-500* 24 20.20 18.70 23.50 0.60 5.00 15.90 0.00 35.70 52.2048 21.40 19.17 22.45 0.66 4.56 16.34 0.29 36.01 53.03 Millet Tg L-500*24 23.50 38.00 16.40 5.00 1.70 8.30 2.50 28.0 43.90 48 23.9 40.20 17.135.36 0.14 6.08 5.49 24.26 41.19 Rice Tg L-500* 24 25.50 15.90 22.90 0.606.80 18.90 1.70 33.20 54.40 48 26.00 17.83 20.30 0.54 4.58 22.13 0.3834.21 51.25

As shown in Table 9, IMO Numbers of 41 to 54% (52.20, 53.03, 43.90,41.19, 54.40, and 51.25) were achieved.

Example 9 Mixed Grain/Cereals Composition

The data in Example 5 for wheat and Example 6 for barley and rye showedconsiderable amount of endogeneous maltogenic enzyme activity resultingin a syrup containing very high maltose. On the other hand, grains knownto not contain endogenous maltogenic enzymes, for example sorghum,millet and rice, required the addition of exogenous maltogenic enzymefor producing the substrate suitable for transglucosidase treatment. Inthis experiment we studied the supplementation of maltogenic enzymecontaining cereals like wheat or barley with sorghum and rice forconverting the starch to substrates containing high maltose levels. In atypical experiment, a 15% suspension of sorghum and rice was prepared bysuspending 140 grams of the flour in 720 grams of deionized water. ThepH was adjusted to pH 5.5 using 6 N H₂SO₄ and Bacillusstearothermophilus alpha amylase sold under the trademark “GC007”(Genencor International) was added at 0.5 kg/mt of the flour. Theresultant suspension was then left in a water bath maintained at 75° C.The suspension was stirred continuously for 6 hours. The temperature wasthen reduced to 60° C. Solid content of flour, e.g., pre-treated riceflour, was increased from 15% to 30% by the addition of barley flour.Similarly, wheat was added to pre-treated sorghum to a finalconcentration to reach 30%. The incubation was then continued for anadditional 10-12 hrs. at 60° C. The pH was reduced to 4.5 andTRANSGLUCOSIDASE L-500 was added at 1.0 kg/mt flour. The incubation at60° C. was continued for 24 hours and 48 hours. The samples were takenfor HPLC analysis and brix; the results are shown in Table 10.

TABLE 10 Soluble Carbohydrate Composition of Millet and Barley; and Riceand Wheat Time Branch Grains/Cereals (hours) Brix IMO No Glucose MaltoseIsomaltose Panose Maltotriose Isomaltotriose G3+ Wheat & 24 16.1 48.7029.50 18.60 2.90 1.00 13.00 3.40 29.50 Sorghum (50:50) 48 16.5 45.2734.68 17.09 5.03 0.51 6.20 6.42 27.63 Rice & Barley 24 21.00 51.00 19.5018.40 1.00 4.20 20.20 1.10 34.7 (50:50) 48 21.4 56.20 21.60 18.21 1.32.92 17.96 2.08 37.86

As shown in Table 10 above, the mixtures of millet and barley; and riceand wheat resulted in 45 to 56% IMO in the resultant suspension afterthe above described incubation periods.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method for making an isomalto-oligosaccharide grain compositionsaid method comprising: (a) contacting an ungelatinized starch in grain(insoluble starch) with an exogenous maltogenic enzyme and a starchliquefying enzyme to produce maltose; (b) contacting said maltose with atransglucosidic enzyme, wherein said steps (a) and (b) occur at atemperature less than or at the gelatinization temperature of saidstarch; and (c) obtaining a grain composition having an enzymaticallyproduced isomalto-oligosaccharide, wherein said oligosaccharide isobtained from said grain.
 2. The method according to claim 1, whereinsaid steps (a) and (b) occur concurrently.
 3. The method according toclaim 1, further comprising the step of drying saidisomalto-oligosaccharide with or without separating insoluble solids. 4.The method according to claim 1, wherein said grain is selected from thegroup consisting of wheat, rye, barley, and malt.
 5. The methodaccording to claim 1, wherein said grain is selected from the groupconsisting of millet, sorghum and rice.
 6. The method according to claim1, wherein said maltogenic enzyme is a beta amylase.
 7. The method ofclaim 6, wherein said beta amylase is obtained from a fungal, bacterial,or plant source.
 8. The method of claim 6, wherein said beta amylase isobtained from a Bacillus species.
 9. The method of claim 8, wherein saidBacillus species is selected from Bacillus stearothermophilus, Bacillusamyloliquefaciens, and Bacillus licheniformis.
 10. The method of claim6, wherein said beta amylase is obtained from a plant source selectedfrom soybean, wheat, and barley.
 11. The method according to claim 1,wherein said starch liquefying enzyme is an alpha amylase obtained froma Bacillus species.
 12. The method according to claim 11, wherein saidBacillus species is Bacillus licheniformis or Bacillusstearothermophilus.
 13. The method according to claim 1, wherein saidtransglucosidic enzyme is a transglucosidase.
 14. The method accordingto claim 13, wherein said transglucosidase is obtained from anAspergillus species.
 15. The method according to claim 14, wherein saidAspergillus species is Aspergillus niger.
 16. The method according toclaim 1, wherein said isomalto-oligosaccharide is further purified. 17.The method of claim 1, wherein said isomalto-oligosaccharide is used asa food additive.
 18. The method of claim 1, wherein saidisomalto-oligosaccharide is used in a flour composition.
 19. The methodof claim 1, wherein said isomalto-oligosaccharide is used in an oralrehydration solution.
 20. The method of claim 1, wherein saidtemperature less than or at the starch gelatinization temperature isbelow 50° C. to 70° C.
 21. The method of claim 1, wherein saidtemperature less than or at the starch gelatinization temperature isbelow 55° C. to 65° C.
 22. The method of claim 1, wherein saidtemperature less than or at the starch gelatinization temperature isbelow 60° C.
 23. The method of claim 1, wherein step (a) furthercomprises contacting said ungelatinized starch with a debranchingenzyme.
 24. The method of claim 23, wherein said debranching enzyme is apullulanase enzyme.